Project Summary The accurate replication and proper division of the genome is essential to life, and ?checkpoints? are placed throughout the cell cycle to ensure accuracy. Much less is known about the presence of checkpoints for the inheritance of functionally correct cytoplasmic organelles. The ER, a large and essential organelle, generates virtually all secretory and transmembrane proteins, and most lipids of the cell. We previously discovered a cell cycle checkpoint, the ER Stress Surveillance pathway (ERSU), which is vital for the inheritance of functionally correct ER by daughter cells in the model organism S. cerevisiae. When activated by ER stress, the ERSU pathway: (1) blocks the inheritance of damaged ER by preventing the ?initiating? ER tubule from entering the daughter cell, (2) mislocalizes the septin ring from the site of cytokinesis, and ultimately, (3) leads to a cell cycle arrest until a functional ER can be re- established. Cells that are mutant in the ERSU pathway die upon ER stress, underscoring the importance of the ERSU. Notably, the ERSU pathway is distinct from the well known Unfolded Protein Response (UPR). In the past grant period, in search of the signal that activates ERSU, we found that phytosphingosine (PHS), an early intermediate of sphingolipid biosynthesis, is an ERSU activator. PHS increases upon ER stress induction and, when exogenously added, PHS sets in motion all the ERSU hallmark events. Moreover, we identified novel transmembrane domain mutants in Reticulon 1 (Rtn1), a protein important for correct ER structure: these mutants inactivate ERSU without affecting overall ER structure. In AIM 1, we will investigate how phytosphingosine activates the ERSU pathway. We also will test how Reticulons are altered by PHS to prevent damaged ER inheritance. In AIM 2, we will investigate how ER tubule inheritance is blocked at the molecular level, examining the role of key cell cycle structures such as septins, cytoskeletal elements, the exocyst and polarisome. In AIM 3, we will: (A) Address whether there is a mammalian ERSU pathway. Our observations of mislocalization of septins upon mammalian ER stress provide initial compelling evidence. (B) Interrogate the impact of ER stress on the mammalian cell cycle including major mitotic cell cycle structural changes that involve the ER, such as ER clearing, nuclear disassembly and reassembly. Understanding the mechanisms by which the ER is inherited in normal cells and how this is perturbed under stress conditions will contribute to our understanding of human disease. Dysregulated ER function is a prominent feature of diabetes, Alzheimer's and Parkinson's, key public health concerns. We hope our study will point towards new treatments for such diseases.